JP2815461B2 - Neurotrophic activity inhibitor - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel substance having an inhibitory effect on neurotrophic activity. The substance of the present invention is effective for treating Alzheimer's disease.

[Prior Art] Alzheimer's disease is an organic dementia usually occurring in people aged 50 years or older, and may be accompanied by Alzheimer's sclerosis, neurofibrillary degeneration, senile plaques and the like. Alzheimer's disease is thought to be associated with enhanced neuronal metabolism and abnormal regeneration, but no effective treatment has been found.

[Problem to be Solved by the Invention] Accordingly, an object of the present invention is to provide a novel neurotrophic activity inhibitor which is effective for treating Alzheimer's disease.

[Means for Solving the Problems] As a result of earnest studies, the present inventors have found that a completely novel protein that is present in the brain of Alzheimer's disease patients but not present in the brain of normal elderly people. The present inventors have found a substance having an effect of suppressing neurotrophic activity and completed the present invention.

That is, the present invention provides a neurotrophic activity inhibitor, which is a protein extracted from human brain tissue and having a neurotrophic activity inhibitory action.

[Effect of the Invention] According to the present invention, a novel neurotrophic activity inhibitor is provided. The novel substance of the present invention is thought to play an important role in preventing the metabolism and abnormal regeneration of neurons,
For example, great clinical effects are expected for Alzheimer's disease patients.

[Specific description of the invention] As described above, the substance of the present invention is a protein extracted from human brain and having an action of suppressing neurotrophic activity. The neurotrophic activity is a substance produced in the hippocampus or cerebral cortex and is greatly involved in maintaining the survival of neurons. Specifically, it can be measured by the method described in Examples below. As described above, the substance of the present invention is a completely novel protein that is present in the brain of a normal elderly person but is not present in the brain of an Alzheimer's disease patient.

The substances of the present invention obtained in the following examples have the following properties.

(1) Molecular weight: about 5000 (SD described in detail in Examples below)
(Measured by S-polyacrylamide gel electrophoresis) (2) Property: White amorphous powder (3) Stable pH range: Stable at pH 3.0 to 7.7 (4) Thermal stability: Insulation at 37 ° C for 20 hours or 100 ° C For 5 minutes to maintain the effect of suppressing neurotrophic activity.

Has a partial amino acid sequence of

The substance of the present invention is a set of two or more kinds of operations of salting out, ultrafiltration, ion exchange chromatography, gel filtration, or high performance liquid chromatography, as it is or after concentrating an extract extracted from human brain tissue. It can be purified together, and the specific method is shown in the following examples.

Hereinafter, the present invention will be described more specifically based on examples.

Example 1 Separation and purification of this substance To 20 g of gray matter of normal human cerebral cortex, 60 ml of water was added, homogenized, and centrifuged at 20,000 g for 1 hour.
5 ml were obtained.

Ultrafiltration was performed on 55 ml of the obtained supernatant using an Amicon YM-10 membrane (trade name), and a fraction having a molecular weight of 10 kDa or more was subjected to DE filtration.
Place on an AE-Sephacel column (1.6 cmφ × 16 cm, manufactured by Pharmacia) and wash buffer (50 mM NaCl, 20 mM Tris
-Cl (pH 7.6)), and extracted with 320 ml of 20 mM Tris-Cl (pH 7.6) buffer with a linear gradient of 50 mM to 300 mM NaCl. FIG. 1 shows a chromatogram obtained by the DEAE-Sephacel column. Fraction No.31
Fractions having inhibitory activities from to to 38 were collected (40 ml), dialyzed, concentrated using Ficoll 400, and then subjected to gel filtration (column size 7.5 mmφ × 6 cm) using TSK G2000SW (manufactured by Tosoh Corporation),
The active fractions of fraction Nos. 30 to 32 were collected (2.5 ml),
It was dialyzed in 5 mM phosphate buffer (pH 7.4). TSK above
FIG. 2 shows the results of gel filtration chromatography using G2000SW. After concentrating the liquid volume to 550 μl, C18 reverse phase HPLC
It was applied to a column (4.6 mmφ × 25 cm, manufactured by Senshu Chemical Company). For elution, a 5 mM ammonium formate solution with a linear gradient of 0% to 80% acetonitrile was used. The result of the C18 reverse phase HPLC chromatography is shown in FIG. Third
As shown, substantially only one sharp peak was obtained by C18 reverse phase HPLC chromatography, indicating that the substance of the present invention was isolated.

Example 2 Measurement of properties The following various properties of the substance obtained in Example 1 were measured.

(1) Ultraviolet Absorption Spectrum An ultraviolet absorption spectrum was measured using a 3 μg distilled aqueous solution of the substance obtained in Example 1 with a spectrophotometer (DU65, manufactured by Beckman). The results are shown in FIG.

(2) Stability The substance obtained in Example 1 was prepared in an aqueous solution of 20 μg / ml, and trifluoroacetic acid was added to 10 μl of the solution to a final concentration of 0.1% (pH 3.0), and added at 37 ° C. for 20 hours. After warming, it was freeze-dried. Dulbecco's phosphate buffer (PBS (-))
In 100 μl of a 2 μg / ml aqueous solution of this substance, and heated at 37 ° C. for 20 hours or 100 ° C. for 5 minutes. Then, using 10 μl of this solution, the inhibitory activity was determined by the method shown in Example 3 below. Was measured, but no decrease in activity-suppressing activity was observed. Further, a 20 μg / ml aqueous solution of the substance obtained in Example 1 was adjusted to pH 7.7 with aqueous ammonia, and a 10 μl thereof was subjected to a stability test in the same manner as described above. As a result, no decrease in the inhibitory activity was observed. Was.

(3) Molecular weight 5 μg of the substance obtained in Example 1 was dissolved in 10 μl of water,
Molecular weight marker (chymotrypsinogen A (molecular weight 250
0), 7.5% to 20% using cytochrome C (molecular weight: 12500), aprotinin (molecular weight: 6500), manufactured by Bio-Rad.
As a result of measurement by SDS-polyacrylamide gel electrophoresis having a concentration gradient of%, it was confirmed that the molecular weight was about 5,000 daltons. FIG. 5 shows the results of the electrophoresis.

Example 3 Measurement of Neurotrophic Activity Inhibitory Activity Cells prepared from cerebral cortex of neonatal rat were placed on a 6 mm microplate coated with geratin-polyoruitin for 1.7 minutes.
× 10 ⁴ cells were seeded, and insulin and transferrin were added to a serum-free medium MEMN2 (Eagle's basic medium) containing 100 μl of an aqueous solution prepared at a concentration of 125 μg / ml of an Alzheimer's disease brain extract obtained in the same manner as in Example 1. , Putrescine, progesterone, and sodium selenite) were added, and 20 ng of the substance obtained in Example 1 was added. The mixture was cultured at 37 ° C. for 5 days in a 5% carbon dioxide gas culture tank. With paraformaldehyde
After immobilization with a 90% methanol / 5% acetic acid solution, the amount of MAP2 was quantified by ELISA using a microtubule-bound protein 2 (MAP2) antibody (Amersham). On the other hand, the amount of MAP2 when the culture was added to the brain extract of Alzheimer's disease without adding this substance was quantified, and the inhibitory activity was expressed by the percentage of decrease in the amount of MAP2.

Using the above method, the relationship between the amount of the substance and the inhibition rate was measured. The results are shown in FIG. As shown in FIG. 6, the inhibitory activity was equilibrated at a concentration of 0.2 μg / ml of the substance, and the inhibitory activity was about 90%.

Example 4 Analysis of Amino Acid Sequence 50 μg of the substance obtained in Example 1 was pyridylethylated by a conventional method, and 0.5 μg of trypsin (manufactured by Sigma) was added to 100 μl of 0.1 M Tris-Cl (pH 8.0) solution. C., reacted for 5 hours, separated by C18 reverse-phase HPLC (0-80% acetonitrile / 0.1% trifluoroacetic acid solution), and analyzed by a protein sequencer (Applied Biosystems, Inc., model 477A). The partial amino acid sequence of this substance was determined by comparing and decoding that of the standard substance with the retention time. As a result, it was found that this substance had the following partial amino acid sequence.

[Brief description of the drawings]

FIG. 1 shows a homogenized normal human cerebral cortex, ultrafiltration, and a caratogram of a fraction having a molecular weight of 10 kDa or more applied to a DEAE-Sephacel column. FIG. 2 shows a purification process of the substance of the present invention. FIG. 3 shows a chromatogram obtained by subjecting a fraction having inhibitory activity to gel filtration, FIG. 3 shows a chromatogram obtained by subjecting the substance of the present invention to C18 reverse phase HPLC, FIG. 4 shows an ultraviolet absorption spectrum of the substance of the present invention, and FIG. FIG. 6 shows an SDS-PAGE electrophoresis pattern of the substance of the present invention. FIG. 6 is a view showing the relationship between the amount of the substance of the present invention and the inhibition rate.

Claims (2)

(57) [Claims] 1. A protein extracted from brain tissue and having a neurotrophic activity-suppressing action, having the following partial amino acid sequence (1) and physicochemical properties (2). (2) Molecular weight: about 5000 Properties: White amorphous powder pH stability: Stable at pH 3.0 to 7.7 Thermal stability: Stable even when kept at 37 ° C for 20 hours or heated at 100 ° C for 5 minutes 2. A therapeutic agent for Alzheimer's disease, comprising the substance having an inhibitory effect on neurotrophic activity according to claim 1.